Introduction: The influenza A virus nonstructural protein NS1 is crucial for the virus infection process. By interacting with various host RNAs or proteins, NS1 regulates both host and viral activities. Nucleolar and coiled-body phosphoprotein 1 (NOLC1), a protein involved in cell cycle regulation, may be involved in host viral resistance. The purpose of this study was to elucidate the changes in host NOLC1 during virus infection and the effect of these changes on virviral replication. Methods: We used co-immunoprecipitation to confirm that H1N1 NS1 can interact with NOLC1 in host cells. Results: We found that the D125 and R200 residues in H1N1 NS1 are crucial for the interaction. Real-time PCR and western blotting revealed changes in host NOLC1 during viral infection. Constructing a lentiviral vector, and then overexpressing and knocking down NOLC1 in A549 cells, enabled us to demonstrate how NOLC1 affects viral replication. We found that H1N1 NS1 interacts with host NOLC1 and that NOLC1 is inhibited during H1N1 infection. In NOLC1-knockdown A549 cells, viral replication was inhibited. Conclusion: The current study revealed that viral replication is negatively affected by a decrease in NOLC1 level in host cells. This research lays the foundation for a deeper comprehension of the mechanisms underlying the complex host–influenza virus interactions, as well as for the prevention and management of influenza viruses.